KR101314880B1 - Rheumatoid arthritis treatment agent - Google Patents

Abstract

Proper combination of alteration of CDR region amino acid sequence, alteration of variable region amino acid sequence, and alteration of normal region amino acid sequence provides better antigen neutralization, pharmacokinetics (plasma retention), immunogenicity, safety, and physical properties than TOCILIZUMAB. Successful creation of a therapeutic agent for arthritis rheumatoid (rheumatic arthritis), pediatric chronic arthritis, or Castleman's disease using the IL-6 receptor antibody as an active ingredient.

Description

Rheumatoid arthritis treatment agent

The present invention relates to a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing an anti-IL-6 receptor antibody as an active ingredient.

IL-6 is a cytokine with various functions, and is produced from various types of cells such as T cells, B cells, monocytes, fibroblasts, osteoblasts, keratinocytes, endothelial cells, mesogenic cells, and synovial cells. Patent document 1, 2). IL-6 binds to the IL-6 receptor, and the signal is delivered in the cell by binding the IL-6 / IL-6 receptor complex to gp130 (Non-Patent Document 3). There are two types of IL-6 receptors, a membrane type and a soluble type, but the soluble IL-6 receptor can also form an IL-6 / IL-6 receptor complex, which can bind to gp130 to deliver a signal.

Arthritis Rheumatism (RA) is a systemic inflammatory disease of unknown origin, in which multiple arthritis and progressive joint destruction are the main symptoms, and lesions spread to the lungs, kidneys, subcutaneous tissues, and the like as extra-articular symptoms. Although various systemic signs exist, the essence of RA is persistent synoviitis, which is characteristic of the disease: synoviitis and subsequent destruction of joint function, which cause cartilage destruction and bone erosion. In the case of RA, the blood vessels of the joint increase, and leukocytes such as lymphocytes and macrophages flow into the synovial tissue from the blood vessels. An immune response occurs in the joint area, an inflammatory reaction is induced by the action of cytokines produced by lymphocytes or macrophages, and cartilage and bone destruction progress.

In the case of RA, hyperacupuncture, an increase in CRP, an increase in platelet count, an increase in polyclonal immunoglobulin, or a rheumatoid factor have been identified, suggesting the involvement of IL-6 in these. It is reported that the IL-6 concentration in the serum and joint fluid of RA patients showed a high value, and this IL-6 level correlates with the disease activity of RA (Nonpatent Document 4-6). In addition, it has been confirmed that production of IL-6 from synovial tissue is also enhanced (Non Patent Literature 7). Furthermore, it has been reported that IL-6 promotes differentiation of osteoclast progenitor cells into osteoclasts in the presence of soluble IL-6 receptors and is also involved in bone resorption (Non Patent Literature 8). These suggest that IL-6 and soluble IL-6 receptors are involved in joint destruction, and indeed, soluble IL-6 receptor concentrations are also high in the joint fluid of RA patients, including IL-6. The concentration is sufficient to induce osteoclasts in vitro (Non-Patent Document 9). As mentioned above, it is thought that IL-6 is involved in various actions, such as antibody production, lymphocyte infiltration, pannus formation, joint destruction, acute reaction, and anemia in the condition of RA (Non-Patent Document 10). ). Recently, excellent clinical trials of RA of humanized anti-IL-6R antibody TOCILIZUMAB, which can bind to both cellular IL-6R and soluble IL-6 receptors and inhibit IL-6 signals, have been clarified. Inhibition of the IL-6 receptor has been shown to be a very effective means for the treatment of RA (Non-Patent Document 10).

In addition, TOCILIZUMAB is known to be effective in the treatment of pediatric chronic arthritis and Castleman's disease in addition to RA.

Further, prior art document information related to the invention of the present application is shown below.

 Kishimoto T. The biology of interleukin-6. Blood 1989; 74: 1-10.  Guerne PA, Zuraw BL, Vaughan JH, Carson DA, Lotz M. Synovium as a source of interleukin 6 in vitro. Contribution to local and systemic manifestations of arthritis. J Clin Invest 1989; 83: 585-92.  Nishimoto N, Kishimoto T. Interleukin 6: from bench to bedside. Nature Clinical Practice Rheumatology 2006; 11: 19-26.  Hirano T, Matsuda T, Turner M, Miyasaka N, Buchan G, Tang B, Sato K, Shimizu M, Maini R, Feldmann M, Kishimoto T: Excessive production of interleukin 6 / B cell stimulatory factor-2 in rheumatoid arthritis. European Journal of Immunology 1988; 18: 1797-1801.  Houssiau FA, Devogelaer JP, Van Damme J, De Deuxchaisnes CN, Van Snick J: Interleukin-6 in synovial fluid and serum of patients with rheumatoid arthritis and other inflammatory arthritides. Arthritis & Rheumatism. 1988; 31: 784-788.  Madhok R, Crilly A, Watson J, Capell HA. Serum interleukin 6 levels in rheumatoid arthritis: correlations with clinical and laboratory indices of disease activity. Ann Rheum Dis 1993; 52: 232-4.  Sack U, Kinne RW, Marx T, Heppt P, Bender S, Emmrich F: Interleukin-6 in synovial fluid is closely associated with chronic synovitis in rheumatoid arthritis. Rheumatolology International 1993; 13: 45-51.  Tamura T, Udagawa N, Takahashi N, Miyaura C, Tanaka S, Yamada Y, Koishihara Y, Ohsugi Y, Kumaki K, Taga T, Kishimoto T, Suda T: Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6. Proceedings of the National Academy of Sciences of the United States of America USA 1993; 90: 11924-11928.  Kotake S, Sato K, Kim KJ, Takahashi N, Udagawa N, Nakamura I, Yamaguchi A, Kishimoto T, Suda T, Kashiwazaki S: Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation. Journal of Bone & Mineral Research 1996; 11: 88-95.  Inhibiting interleukin-6 in rheumatoid arthritis. Choy E. Curr Rheumatol Rep. 2008 Oct; 10 (5): 413-7.

This invention is made | formed in view of such a situation, and the objective is to provide the therapeutic agent of arthritis, pediatric chronic arthritis, or Castleman's disease containing an anti-IL-6 receptor antibody as an active ingredient. More specifically, by replacing the amino acid of Tocilizumab, which is used as a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease, anti-neutralization, pharmacokinetics (retention in plasma), immunogenicity, safety and physical properties are improved. A therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease using the IL-6 receptor antibody as an active ingredient.

The present inventors have substituted the amino acid of Tocilizumab, which is used as a therapeutic agent for arthritis rheumatoid (rheumatic arthritis), pediatric chronic arthritis or Castleman's disease, and thus the antigen-neutralizing ability, pharmacokinetics (retention in plasma), immunogenicity, safety and physical properties Improved anti-IL-6 receptor antibodies were obtained. And it discovered that the agent containing this anti-IL-6 receptor antibody as an active ingredient is useful for the treatment of arthritis, pediatric chronic arthritis, or Castleman's disease.

More specifically, the present invention provides the following [1] to [3].

[1] a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibodies as an active ingredient;

(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence set forth in CDR3),

(b) CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3), and amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising a CDR3 having a

(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 A heavy chain comprising CDR3 having the amino acid sequence of CDR3), and a CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), a CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3), a sequence An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in No. 6 (CDR3 of VL3).

[2] a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibodies as an active ingredient;

(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),

(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),

(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising.

[3] a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibodies as an active ingredient;

(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),

(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),

(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

Furthermore, this invention provides the following.

[4] a method for treating arthritis, pediatric chronic arthritis or Castleman's disease, comprising the steps of administering the following antibodies;

(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence set forth in CDR3),

(b) CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3), and amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising a CDR3 having a

(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 A heavy chain comprising CDR3 having the amino acid sequence of CDR3), and a CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), a CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3), a sequence An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in No. 6 (CDR3 of VL3).

[5] a method for treating arthritis, pediatric chronic arthritis or Castleman's disease, comprising the steps of administering the following antibodies;

(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),

(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),

(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising.

[6] a method for treating arthritis, pediatric chronic arthritis or Castleman's disease, comprising the steps of administering the following antibodies;

(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),

(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),

(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

[7] use of the following antibodies in the manufacture of a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease;

(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence set forth in CDR3),

(b) CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3), and amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising a CDR3 having a

(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 A heavy chain comprising CDR3 having the amino acid sequence of CDR3), and a CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), a CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3), a sequence An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in No. 6 (CDR3 of VL3).

[8] use of the following antibodies in the manufacture of a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease;

(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),

(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),

(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising.

[9] use of the following antibodies in the preparation of a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease;

(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),

(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),

(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

[10] the following antibodies for use in the method of treating arthritis, pediatric chronic arthritis or Castleman's disease;

(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence set forth in CDR3),

(b) CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3), and amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising a CDR3 having a

(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 A heavy chain comprising CDR3 having the amino acid sequence of CDR3), and a CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), a CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3), a sequence An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in No. 6 (CDR3 of VL3).

[11] the following antibodies for use in the method of treating arthritis, pediatric chronic arthritis or Castleman's disease;

(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),

(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3),

(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising.

[12] the following antibodies for use in the method of treating arthritis, pediatric chronic arthritis or Castleman's disease;

(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),

(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3),

(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

The therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the anti-IL-6 receptor antibody obtained by the present invention as an active ingredient includes antigen-neutralizing ability, pharmacokinetics (retention in plasma), immunogenicity, safety and physical properties. As a result, the frequency of administration can be reduced, and the therapeutic effect can be exerted continuously.

1 is a diagram showing the plasma concentration change of antibodies when TOCILIZUMAB and Fv4-M73 are administered to a cynomolgus monkey at 1 mg / kg.
Fig. 2 is a graph showing the plasma concentration change of CRP when TOCILIZUMAB and Fv4-M73 were administered to crab monkeys at 1 mg / kg.
Fig. 3 is a graph showing plasma concentrations of unbound soluble IL-6 receptor rate when TOCILIZUMAB and Fv4-M73 were administered to crab monkeys at 1 mg / kg.
Figure 4 shows the inhibition of MCP-1 production from synovial cells derived from human RA patients by TOCILIZUMAB and Fv4-M73.
Fig. 5 shows the inhibition of VEGF production from synovial cells derived from human RA patients by TOCILIZUMAB and Fv4-M73.

The present invention relates to a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibodies as an active ingredient.

(a) a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73),

A CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73),

CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (CDR3 of VH3-M73)

An antibody comprising a heavy chain comprising a

(b) a CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3),

A CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3),

CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3)

An antibody comprising a light chain comprising a, or

(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73),

A CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73),

A heavy chain comprising a CDR3 having the amino acid sequence set forth in SEQ ID NO: 3 (CDR3 of VH3-M73), and

CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3),

A CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3),

An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3).

Moreover, this invention relates to the therapeutic agent of arthritis, pediatric chronic arthritis, or Castleman's disease containing the following antibodies as an active ingredient.

(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),

(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3), or

(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising.

Moreover, this invention relates to the therapeutic agent of arthritis, pediatric chronic arthritis, or Castleman's disease containing the following antibodies as an active ingredient.

(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),

(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3), or

(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

In addition, the present invention, in the antibody of the present invention having the above-described amino acid sequence, 1 or a plurality of amino acids (usually within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, more preferably 3 Within amino acids) to alter (substitute, delete, add and / or insert, etc.) or provide an antibody with a modified amino acid sequence. Antibodies in which these amino acid sequences have been modified or modified preferably have the same activity (antigen binding activity, antigen neutralizing activity, etc.) as the original antibody.

In addition, the bispecific antibody may be sufficient as the antibody used by the antibody of this invention. A bispecific antibody refers to an antibody having variable regions in the same antibody molecule that recognize different epitopes. In the present invention, the bispecific antibody may be a bispecific antibody that recognizes different epitopes on the IL-6 receptor molecule, one antigen-binding site recognizes IL-6 receptor, and the other antigen-binding site is different. It is also possible to set it as a bispecific antibody which recognizes. As an antigen to which the other antigen binding site of the bispecific antibody which becomes the antibody of this invention which recognizes an IL-6 receptor binds, for example, IL-6, TNF (alpha), TNFR1, TNFR2, CD80, CD86, CD28, CD20 , CD19, IL-1α, IL-β, IL-1R, RANKL, RANK, IL-17, IL-17R, IL-23, IL-23R, IL-15, IL-15R, BlyS, lymphotoxinα, lymphotoxinβ, LIGHT ligand, LIGHT, VLA-4, CD25, IL-12, IL-12R, CD40, CD40L, BAFF, CD52, CD22, IL-32, IL-21, IL-21R, GM-CSF, GM-CSFR, M- CSF, M-CSFR, IFN-alpha, VEGF, VEGFR, EGF, EGFR, CCR5, APRIL, APRILR.

FR used for the antibody of this invention is not specifically limited, A person skilled in the art can select suitably. Although it does not specifically limit, Preferably, FR derived from a human is used. The amino acid may be modified from the natural sequence of FR.

The normal region used for the antibody of the present invention is not particularly limited and can be appropriately selected by those skilled in the art. Although not specifically limited, Preferably, the normal region derived from a human is used. In the normal region, amino acids may be modified from natural sequences.

The antibody of the present invention described above is an anti-IL-6 receptor antibody having excellent antigen neutralizing ability, pharmacokinetics (plasma retention), immunogenicity, safety, and / or physical properties. Very useful as a therapeutic agent.

The antibody of the present invention can be produced by a method known to those skilled in the art.

For example, it is possible to prepare a gene encoding the antibody of the present invention, insert the gene into an appropriate vector, introduce it into a host, and produce it using genetic recombination techniques (for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

Accordingly, the present invention provides a method for producing an antibody of the present invention, comprising the step of culturing a host cell comprising a vector into which the gene encoding the antibody of the present invention is introduced.

More specifically, the manufacturing method of the antibody of this invention containing the following process is provided.

(a) culturing a host cell comprising a vector into which the gene encoding the antibody of the present invention is introduced,

(b) obtaining the antibody encoded by the gene.

Specifically, in the case of production using mammalian cells, it may be expressed by a commercially available useful promoter, an antibody gene to be expressed, a DNA functionally bound to a polyA signal downstream of its 3 'side, or a vector comprising the same. For example, as a promoter / enhancer, human cytomegalovirus immediate early promoter / enhancer is mentioned.

In addition, as a promoter / enhancer that can be used for antibody expression used in the present invention, viral promoters / enhancers such as retrovirus, polyoma virus, adenovirus, monkey virus 40 (SV40), and human extender 1α (HEF1α) It is possible to use a promoter / enhancer derived from a mammalian cell, for example.

For example, when using the SV40 promoter / enhancer, Mulligan et al. (Mulligan, RC et al., Nature (1979) 277, 108-114), and also when using the HEF1α promoter / enhancer. , Mizushima et al. (Mizushima, S. and Nagata, S. Nucleic Acids Res. (1990) 18, 5322) can be easily carried out.

In the case of production using E. coli, commercially available useful promoters, signal sequences for antibody secretion, and the antibody genes to be expressed can be functionally linked and expressed. For example, as a promoter, a lacZ promoter and an araB promoter can be mentioned. When using the lacZ promoter, Ward et al. (Ward, ES et al., Nature (1989) 341, 544-546; Ward, ES et al. FASEB J. (1992) 6, 2422-2427) In the case of using the araB promoter, according to the method of Better et al. (Better, M. et al. Science (1988) 240, 1041-1043).

As a signal sequence for antibody secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379-4383) may be used when produced by E. coli periplasm. After the antibody produced by the periplasm is isolated, the structure of the antibody is appropriately refolded and used (see, for example, WO96 / 30394).

As the origin of replication, those derived from SV40, polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can be used. Also, for amplification of gene copy number in the host cell system, the expression vector is an aminoglycoside as a selection marker. Transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthinguan phosphoribosyl transferase (Ecogpt) gene, dehydrofolate reductase (dhfr) gene and the like.

For the production of the antibodies used in the present invention, any production system can be used.

Production systems for antibody production include production systems in vitro and in vivo. Examples of in vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.

In the case of using eukaryotic cells, there are production systems using animal cells, plant cells, or fungal cells. As animal cells, (1) mammalian cells, such as CHO, COS, myeloma, baby hamster kidney (BHK), HeLa, Vero, etc., (2) amphibian cells, for example, African-clawed oocytes, or ( 3) Insect cells such as sf9, sf21, Tn5 and the like are known. As plant cells, cells derived from Nicotiana tabacum are known and may be callus cultured. As fungal cells, yeast, for example, Saccharomyces, for example Saccharomyces cerevisiae, filamentous fungi, for example Aspergillus, for example Aspergillus niger and the like are known.

In the case of using prokaryotic cells, there is a production system using bacterial cells. As bacterial cells, E. coli and Bacillus subtilis are known.

Antibodies are obtained by introducing a target antibody gene into these cells by transformation and culturing the transformed cells in vitro. Cultivation is performed according to a well-known method. For example, DMEM, MEM, RPMI1640, IMDM can be used as the culture solution, and serum supplements such as fetal bovine serum (FCS) can also be used in combination. Alternatively, the antibody may be produced in vivo by transferring the cells into which the antibody gene has been introduced to the animal's abdominal cavity or the like.

On the other hand, examples of production systems of in vivo include production systems using animals and production systems using plants. In the case of using animals, there are mammalian animals and production systems using insects.

As mammals, goats, pigs, sheep, mice, cows and the like can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). As an insect, silkworms can be used. In the case of using plants, for example, tobacco can be used.

Antibody genes are introduced into these animals or plants, and antibodies are produced and recovered in the body of the animals or plants. For example, an antibody gene is inserted in the middle of a gene encoding a protein inherently produced in milk such as goat β casein to prepare as a fusion gene. The DNA fragment containing the fusion gene into which the antibody gene was inserted is injected into a goat's embryo, and the embryo is introduced into a female goat. The desired antibody is obtained from the milk produced by the transgenic goat or its offspring which are born from the goat which received the embryo. In order to increase the amount of milk containing the desired antibody produced from the transgenic goat, hormones may be appropriately used in the transgenic goat (Ebert, K. M. et al., Bio / Technology (1994) 12, 699-702).

When silkworms are used, baculoviruses in which the target antibody genes are inserted are infected with silkworms, and the target antibodies are obtained from the fluids of these silkworms (Maeda, S. et al., Nature (1985) 315, 592. -594). In the case of using tobacco, a target antibody gene is inserted into a plant expression vector, such as pMON530, and the vector is introduced into a bacterium such as Agrobacterium tumefaciens. The bacteria are infected with tobacco, for example Nicotiana tabacum, and the desired antibody is obtained from the leaves of the tobacco (Julian, K.-C. Ma et al., Eur. J. Immunol. ( 1994) 24, 131-138).

When the antibody is produced in vitro or in vivo as described above, the DNA encoding the antibody heavy chain (H chain) or light chain (L chain) may be separately inserted into an expression vector to cotransform the host. Alternatively, the host may be transformed by inserting the DNA encoding the H chain and the L chain into a single expression vector (see International Patent Application Publication No. WO 94-11523).

Antibodies produced and expressed as described above can be uniformly purified from the cells and separated from the host. Isolation and purification of the antibody used in the present invention can be carried out by affinity chromatography. As a column used for affinity chromatography, a protein A column and a protein G column are mentioned, for example. As a carrier used for the Protein A column, for example, HyperD, POROS, Sepharose F.F. And the like. In addition, what is necessary is just to use the isolation | separation and purification method used by normal protein, and is not limited at all.

For example, by appropriately selecting and combining chromatography, filters, ultrafiltration, salting out, dialysis, and the like other than the affinity chromatography, the antibody used in the present invention can be separated and purified. As chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, etc. are mentioned, for example. These chromatography can be applied to high performance liquid chromatography (HPLC). In addition, reverse phase HPLC may be used.

The concentration measurement of the antibody obtained above can be performed by measurement of absorbance, ELISA, etc. That is, in the case of measurement of absorbance, after dilution with PBS (-) is appropriate, the absorbance at 280 nm is measured, and 1 mg / ml is calculated as 1.35 OD. In addition, in the case of ELISA, it can measure as follows. That is, 100 µl of goat anti-human IgG (manufactured by TAG) diluted to 1 µg / ml in 0.1 M bicarbonate buffer (pH 9.6) was added to a 96-well plate (manufactured by Nunc), and incubated at 4 ° C overnight to incubate the antibody. Make up. After blocking, 100 µl of human IgG (manufactured by CAPPEL) is added as a sample or an article containing the antibody or antibody used in the present invention, which is appropriately diluted, and incubated at room temperature for 1 hour.

After washing, 100 µl of 5000-fold diluted alkaline phosphatase-labeled anti-human IgG (manufactured by BIO SOURCE) is added and incubated at room temperature for 1 hour. After washing, the substrate solution is added and after incubation, the absorbance at 405 nm is measured using MICROPLATE READER Model 3550 (manufactured by Bio-Rad) to calculate the concentration of the target antibody.

The IL-6 signal transduction inhibitory activity of the antibody used by this invention can be evaluated by the method well-known to those skilled in the art used normally. For example, IL-6 dependent human myeloma strain (S6B45, KPMM2), human Linnert T lymphoma cell line KT3, or IL-6 dependent cell MH60.BSF2 are cultured, and IL-6 is added to IL-6 simultaneously. Co-inhibitors can be used to measure ³ H-thymidine uptake of IL-6 dependent cells. In addition, IL-6 receptor-expressing cells The method of culturing U266, and, ¹²⁵ I by the cover at the same time, the addition of IL-6 was added to IL-6 inhibitor, measuring IL-6 ¹²⁵ I labeled IL-6 bound to the receptor expressing cells It may be. In the assay system, the IL-6 inhibitory activity of the IL-6 inhibitor can be evaluated by comparing the results obtained in both groups with a negative control containing no IL-6 inhibitor in addition to the group in which the IL-6 inhibitor is present. .

The antibody to be used in the present invention may be a fragment of the antibody or a modification thereof as long as it can be suitably used in the present invention. For example, as a fragment of an antibody, Fab, F (ab ') 2, Fv, or single chain Fv (scFv), sc (Fv) 2 etc. which connected Fv of H chain and L chain with the appropriate linker are mentioned. .

In addition, the antibody used by this invention can also use the antibody couple | bonded with various molecules, such as polyethyleneglycol (PEG), as a modification of an antibody. "Antibody" as used in the present invention also includes these antibody modifications. In order to obtain such an antibody modified product, it can be obtained by chemically modifying the obtained antibody. These methods have already been established in this field.

Tocilizumab, a humanized anti-human IL-6 receptor antibody, is approved in Japan as a therapeutic drug for arthritis, pediatric chronic arthritis (e.g., acute arthritis of active arthritis, systemic febrile arthritis of arthritis), and Castleman's disease. It is.

The antibody of the present invention described above is an antibody having improved antigen-neutralizing ability, blood dynamics, immunogenicity, safety, and physical properties by modifying the amino acid of Tocilizumab. Therefore, the above-mentioned antibody of the present invention naturally has the same therapeutic effect as that of Tocilizumab, and can be used as a therapeutic agent for arthritis, pediatric chronic arthritis and Castleman's disease.

The therapeutic agent of the present invention can be administered in the form of a medicine, and can be administered orally or parenterally or systemically. For example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, capsular, oral enteric preparation can be selected, and the administration method can be appropriately selected according to the age and symptoms of the patient. . The effective dose is usually selected in the range of 0.01 mg to 100 mg per kg of body weight once. Or, usually, a dosage of 1 to 1000 mg, preferably 50 to 250 mg can be selected per patient. Preferred dosages can also be appropriately selected by those skilled in the art.

In the therapeutic agent of the present invention, a carrier acceptable in formulations such as a preservative or a stabilizer may be added. A carrier acceptable in a formulation may be a material which itself has the above-mentioned therapeutic effect, or may be a material which does not have the above-mentioned therapeutic effect, and means a material which can be administered together with the therapeutic agent. Moreover, the material which does not have a therapeutic effect, or the material which has synergistic or additive stabilizing effect by using together with an antibody may be sufficient.

Examples of materials acceptable in the formulation include sterile water, saline, stabilizers, excipients, buffers, preservatives, surfactants, chelating agents (EDTA, etc.), binders and the like.

In this invention, a nonionic surfactant is mentioned as surfactant, For example, Sorbitan fatty acid ester, such as sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate; Glycerin fatty acid esters such as glycerin monocaprylate, glycerin monomyristate and glycerin monostearate; Polyglycerol fatty acid esters such as decaglyceryl monostearate, decaglyceryl distearate, decaglyceryl monolinoleate; Polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan Polyoxyethylene sorbitan fatty acid esters such as tristearate; Polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate, etc .; Polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glyceryl monostearate; Polyethylene glycol fatty acid esters such as polyethylene glycol distearate; Polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; Polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylene polyoxypropylene glycol, polyoxyethylene polyoxypropylene propyl ether and polyoxyethylene polyoxypropylene cetyl ether; Polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether; Polyoxyethylene hardened castor oil, such as polyoxyethylene castor oil and polyoxyethylene hardened castor oil (polyoxyethylene hydrogen castor oil); Polyoxyethylene beeswax derivatives such as polyoxyethylene sorbet beeswax; Polyoxyethylene lanolin derivatives such as polyoxyethylene lanolin; The thing containing HLB6-18, such as polyoxyethylene fatty acid amides, such as polyoxyethylene stearic acid amide, etc. are mentioned as a typical example.

Moreover, as surfactant, anionic surfactant is also mentioned, For example, Alkyl sulfate which has an alkyl group of 10-18 carbon atoms, such as sodium cetyl sulfate, sodium lauryl sulfate, and sodium oleyl sulfate; Polyoxyethylene alkyl ether sulfates having an average added mole number of ethylene oxide of 2 to 4 and an alkyl group having 10 to 18 carbon atoms, such as sodium polyoxyethylene lauryl sulfate; Alkyl sulfosuccinic acid ester salts having 8 to 18 carbon atoms in an alkyl group, such as sodium lauryl sulfon succinic acid ester; Phospholipids with natural surfactants such as lecithin, glycerol; Sphingophospholipids such as sphingomyelin; Sucrose fatty acid ester of a C12-C18 fatty acid, etc. are mentioned as a typical example.

To the therapeutic agent of the present invention, one kind or a combination of two or more kinds of these surfactants can be added. Preferred surfactants for use in the formulations of the present invention are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60 or 80, with polysorbates 20 and 80 being particularly preferred. Furthermore, polyoxyethylene polyoxypropylene glycol represented by poloxamer (Pluronic F-68 (trademark) etc.) is also preferable.

The amount of the surfactant added varies depending on the type of surfactant used, but in the case of polysorbate 20 or polysorbate 80 or poloxamer 188, the amount is generally 0.0001 to 10% (w / v), preferably It is 0.001 to 5%, More preferably, it is 0.005 to 3%.

In the present invention, as a buffer, other organic acids such as phosphoric acid, citric acid buffer, acetic acid, malic acid, tartaric acid, succinic acid, lactic acid, potassium phosphate, gluconic acid, caprylic acid, deoxycholic acid, salicylic acid, triethanolamine, fumaric acid, and the like, or carbonate Buffers, tris buffers, histidine buffers, imidazole buffers, and the like.

In addition, the solution preparation may be prepared by dissolving in an aqueous buffer solution known in the field of solution preparation. The concentration of the buffer is generally 1 to 500 mM, preferably 5 to 100 mM, and more preferably 10 to 20 mM.

The therapeutic agent of the present invention may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulins, sugars such as amino acids, polysaccharides and monosaccharides, carbohydrates and sugar alcohols.

In the present invention, the amino acid may be a basic amino acid such as arginine, lysine, histidine, ornithine, or an inorganic salt of these amino acids (preferably in the form of hydrochloride, phosphate salt, that is, phosphate amino acid). When free amino acids are used, preferred pH values are adjusted by the addition of suitable physiologically acceptable buffers such as inorganic acids, in particular hydrochloric acid, phosphoric acid, sulfuric acid, acetic acid, formic acid or these salts. In this case, the use of phosphate is particularly advantageous in that a stable freeze-dried product is obtained. When the preparation is substantially free of organic acids such as malic acid, tartaric acid, citric acid, succinic acid, fumaric acid, or the like, or there are no corresponding anions (malic acid ion, tartaric acid ion, citric acid ion, succinic acid ion, fumaric acid ion, etc.) In this case, it is particularly advantageous. Preferred amino acids are arginine, lysine, histidine, or ornithine. In addition, acidic amino acids such as glutamic acid and aspartic acid, and salts thereof (preferably sodium salts) or neutral amino acids such as isoleucine, leucine, glycine, serine, threonine, valine, methionine, cysteine, or alanine, Alternatively, it is also possible to use aromatic amino acids such as phenylalanine, tyrosine, tryptophan, or N-acetyl tryptophan of derivatives.

In the present invention, examples of the sugars and carbohydrates such as polysaccharides and monosaccharides include dextran, glucose, fructose, lactose, xylose, mannose, maltose, sucrose, trehalose, and raffinose. Can be.

In the present invention, examples of sugar alcohols include mannitol, sorbitol, inositol, and the like.

When the agent of the present invention is used as an aqueous solution for injection, it may be mixed with isotonic solution containing, for example, saline solution, glucose or other auxiliary agent (for example, D-sorbitol, D-mannose, D-mannitol, sodium chloride). Can be. The aqueous solution may be used in combination with a suitable dissolution aid (for example, alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50, etc.).

As desired, it may further contain a diluent, a dissolution aid, a pH adjuster, a passivating agent, a sulfur-containing reducing agent, an antioxidant, and the like.

In the present invention, as the sulfur-containing reducing agent, for example, N-acetylcysteine, N-acetyl homocysteine, thioctic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate And a sulfhydryl group such as thioalkanoic acid having 1 to 7 carbon atoms and glutathione.

In the present invention, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and salts thereof, and L-ascorbic acid Chelating agents such as palmitate, L-ascorbic acid stearate, sodium bisulfite, sodium sulfite, molar tristriamyl, molar trispropyl or ethylenediamine disodium acetate (EDTA), sodium pyrophosphate, sodium metaphosphate, and the like. .

Also, if necessary, it is enclosed in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]), or colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles and nanoparticles). Capsules, etc.) (see "Remington's Pharmaceutical Science 16 ^th edition", Oslo Ed., 1980, etc.). In addition, since the method of using a drug as a sustained-release drug is known, it can be applied to the present invention (Langer et al., J. Biomed. Mater. Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982) 12: 98-105; US Patent No. 3,773,919; European Patent Application Publication No. 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; EP No. 133,988). It is also possible to increase the amount of liquid administered subcutaneously by adding or mixing hyaluronidase to the present drug (for example, WO2004 / 078140, etc.).

The formulationly acceptable carrier to be used is selected appropriately or in combination of the above depending on the dosage form, but is not limited thereto.

The present invention relates to a method for treating arthritis, pediatric chronic arthritis, and Castleman's disease, comprising the step of administering a therapeutic agent of the present invention to a subject. In the present invention, the "subject" refers to an organism to which the therapeutic agent of the present invention is administered and a part of the organism. The organisms include, but are not particularly limited to, animals (eg, humans, animal species of animals, wild animals). It does not specifically limit about said "part in a living body".

In the present invention, "administering" includes administration orally or parenterally. Examples of oral administration include administration in the form of oral preparations. As oral preparations, formulations such as granules, powders, tablets, capsules, solvents, emulsions, or suspensions can be selected.

As parenteral administration, administration in the form of injection is mentioned, and intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, etc. are mentioned as injection. In addition, the effect of the method of the present invention can be achieved by introducing a gene containing an oligonucleotide to be administered into a living body using a technique of gene therapy. It is also possible to administer the agent of the present invention locally to the area to be treated. For example, administration may be by topical infusion during surgery, use of a catheter, or targeted gene delivery of DNA encoding the peptides of the invention.

The administration of the therapeutic agent of the present invention may be after a symptom of a disease appears or may be prophylactically administered before the symptom appears.

Moreover, this invention relates to the use of the antibody of this invention in manufacture of the therapeutic agent of arthritis, pediatric chronic arthritis, or Castleman's disease. The present invention also relates to an antibody of the present invention for use in a method of treating arthritis, pediatric chronic arthritis or Castleman's disease.

In addition, when an amino acid included in the amino acid sequence described in the present invention receives a modification (for example, modification of pyroglutamic acid by pyroglutamylation of N-terminal glutamine is a modification well known to those skilled in the art) However, even if such an amino acid is modified after translation, it is naturally included in the amino acid sequence described in the present invention.

Moreover, what kind of structure may be sufficient as the structure of the sugar chain which couple | bonds. The 297th sugar chain of the EU numbering may have any sugar chain structure (preferably fucosylated sugar chain), and the sugar chain may not be bound (for example, produced by E. coli, or the sugar chain is bound to 297th of the EU numbering). By modifying it to avoid).

In addition, all the prior art documents referred in this specification are contained in this specification as a reference.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Monkey PK / PD Test of Anti-Human IL-6 Receptor Antibody

TOCILIZUMAB (H chain WT-IgG1 / SEQ ID NO: 13, L chain WT-kappa / SEQ ID NO: 14), and amino acids in TOCILIZUMAB for the purpose of improving antigen neutralization, blood dynamics, immunogenicity, safety and physical properties of TOCILIZUMAB. Fv4-M73 (H chain VH3-M73 / SEQ ID NO: 9, L chain VL3-kappa / SEQ ID NO: 10) having a substitution or the like was expressed and purified by a method well known to those skilled in the art (see for reference, for a method). The effect as a therapeutic drug of the arthritis rheumatoid was examined as follows.

TOCILIZUMAB and Fv4-M73 were administered to the cynomolgus monkey at 1 mg / kg once intravenously and the plasma concentrations were evaluated (see Reference Example for method). The plasma concentrations after intravenous administration of TOCILIZUMAB and Fv4-M73 are shown in FIG. 1. As a result, Fv4-M73 significantly improved pharmacokinetics in cynomolgus monkeys compared to TOCILIZUMAB.

To evaluate the efficacy of neutralizing the cynomolgus monkey membrane IL-6 receptor, 5 µg / kg of cynomolgus monkey IL-6 from day 6 to day 18 (day 3 to day 10 for TOCILIZUMAB) Was administered subcutaneously to the urinary tract every day, and the CRP concentration of each individual after 24 hours was measured (see Reference Example for the method). The CRP concentration at the time of administration of each antibody is shown in FIG. To assess the effect of neutralizing the cynomolgus soluble IL-6 receptor, the concentration of the non-binding cynomolgus soluble IL-6 receptor in the cynomolgus monkey plasma was measured to determine the non-binding soluble IL-6 receptor. The -6 receptor rate was calculated (see reference example for method). The change of the unbound soluble IL-6 receptor rate at the time of administering each antibody is shown in FIG.

Fv4-M73 more consistently neutralized the cynomolgus macaque IL-6 receptor compared to TOCILIZUMAB, resulting in long-term inhibition of the increase in CRP. In addition, Fv4-M73 more continuously neutralized the cynomolgus monkey soluble IL-6 receptor as compared to TOCILIZUMAB, and inhibited the increase of the non-binding cynomolgus soluble IL-6 receptor for a long time. From this, Fv4-M73 was found to be superior to TOCILIZUMAB in terms of the neutralization persistence of the membrane-type IL-6 receptor and the soluble IL-6 receptor.

EXAMPLE 2

Monocyte chemoattractant protein (MCP) -1 is known to be involved in cell infiltration of monocytes, T cells, NK cells and basophil. It has been reported that MCP-1 is highly expressed in synovial tissue and synovial fluid of RA patients (J Clin Invest. 1992 Sep; 90 (3): 772-9), and is thought to be involved in the condition of RA ( Inflamm Allergy Drug Targets. 2008 Mar; 7 (1): 53-66.).

In addition, VEGF is a potent angiogenesis factor and is known to be produced from macrophages, fibroblasts, synovial cells, and the like in the synovial membrane of RA patients (J Rheumatol. 1995 Sep; 22 (9): 1624-30.). In addition, VEGF levels in the serum of RA patients correlated with disease activity and radiographic progression (Arthritis Rheum. 2003 Jun; 48 (6): 1521-9., Arthritis Rheum. 2001 Sep; 44 (9): 2055-64.) It is thought that VEGF plays an important role in the pathogenesis of RA because RA levels are lowered by treating RA patients with anti-IL-6R antibody TOCILIZUMAB (Mod Rheumatol. 2009; 19 (1)). : 12-9., Mediators Inflamm. 2008; 2008: 129873).

Therefore, TOCILIZUMAB and Fv4-M73 examined whether the production of MCP-1 and VEGF from sIL-6R and IL-6 stimulation from synovial cells derived from human RA patients can be inhibited by the following method.

Human RA patient-derived synovial cells (TOYOBO) are seeded in 96-well plates at 2 × 10 ⁴ /0.05 mL / well in IMDM medium containing 5% FCS and left for 90 minutes in a CO ₂ incubator (37 ° C., 5% CO ₂ ). It was. 0.05 mL of appropriately diluted concentrations of TOCILIZUMAB and Fv4-M73 are added, and after 15 minutes of standing, 0.05 mL of a soluble IL-6 receptor (SR344: prepared according to the reference example) is added, and further left for 30 minutes, and further IL- 0.05 mL of 6 (TORAY) was added (final concentrations of soluble IL-6 receptor and IL-6 were 50 ng / mL each). After 2 days of culture, the culture supernatant was recovered, and the concentration of MCP-1 and VEGF in the culture supernatant was measured using an ELISA kit (Biosource and Pierce Biotechnology). The results are shown in FIGS. 4 and 5. TOCILIZUMAB and Fv4-M73 concentration-dependently inhibited MCP-1 and VEGF production from human RA patient derived synovial cells by soluble IL-6 receptor and IL-6 stimulation.

 From these facts, Fv4-M73 acts as an anti-IL-6 receptor neutralizing antibody (which binds to the IL-6 receptor and blocks the signaling of the membrane-like and soluble IL-6 receptors), compared to TOCILIZUMAB. Excellent, it is possible to significantly reduce the frequency and dosage of administration compared to TOCILIZUMA, and Fv4-M73 inhibits the production of MCP-1 and VEGF from synovial cells derived from human RA patients. It has been shown to be a very useful therapeutic agent.

<Reference example>

Preparation of recombinant soluble human IL-6 receptor

Preparation of human IL-6 receptor as an antigen A soluble human IL-6 receptor was prepared as follows. J. Biochem. Soluble human IL-6 receptor (Namasaki et al., Science 1988; 241: 825-828 (GenBank # X12830)) consisting of the N-terminal first to 344 amino acid sequences reported in 108, 673-676 (1990). CHO cell normal expression strains were prepared. From culture supernatants obtained from SR344 expressing CHO cells, soluble humans were analyzed by Blue Sepharose 6 FF column chromatography, affinity chromatography by a column immobilized with a specific antibody against SR344, and three column chromatography of gel filtration column chromatography. IL-6 receptor was purified. The fraction eluted as the main peak was used as the final product.

Preparation of recombinant soluble goat monkey IL-6 receptor (cIL-6R)

Oligo DNA primers were prepared based on the published Rhesus monkey IL-6 receptor gene sequence (Birney et al, Ensembl 2006, Nucleic Acids Res. 2006 Jan 1; 34 (Database issue): D556-61.) A DNA fragment encoding the full length of the cynomolgus IL-6 receptor gene was prepared by PCR using a primer as a template and cDNA prepared from the cynomolgus monkey pancreas. The obtained DNA fragment was inserted into an animal cell expression vector, and a CHO normal expression line (cyno. SIL-6R producing CHO cell) was produced using this. cyno. Cultures of sIL-6R producing CHO cells were purified by HisTrap column (GE Healthcare Biosciences), concentrated using Amicon Ultra-15 Ultracel-10k (Millipore), and superdex200pg16 / 60 gel filtration column (GE Healthcare Biosciences). ) Was further purified to obtain a final product of the soluble cynomolgus monkey IL-6 receptor (hereinafter, cIL-6R).

Preparation of Recombinant Crab Monkey IL-6 (cIL-6)

Crab monkey IL-6 was prepared as follows. A base sequence encoding the 212 amino acid registered in SWISSPROT Accession No. P79341 was prepared, cloned into an animal cell expression vector, and introduced into CHO cells to prepare a normal expression cell line (cyno. IL-6 producing CHO cells). cyno. Cultures of IL-6 producing CHO cells were purified by SP-Sepharose / FF column (GE Healthcare Bioscience), concentrated using Amicon Ultra-15 Ultracel-5k (Millipore), and superdex 75pg 26/60 gel filtration column (GE). Healthcare bioscience) was further purified, concentrated using Amicon Ultra-15 Ultracel-5k (Millipore) to obtain the final product of cynomolgus monkey IL-6 (hereafter cIL-6).

Production, Expression, and Refining of Variants of TOCILIZUMAB

The plasmid fragment encoding the target antibody sequence was inserted into the animal cell expression vector to prepare the target H chain expression vector and L chain expression vector. The base sequence of the obtained expression vector was determined by a method known in the art. Expression of the antibody was performed using the following method. Human fetal kidney cancer cell-derived HEK293H strain (Invitrogen) was suspended in DMEM medium (Invitrogen) containing 10% Fetal Bovine Serum (Invitrogen), and a dish for adhesion cells at a cell density of 5-6 × 10 ⁵ cells / mL ( After sowing 10 mL with each dish 10 cm in diameter and CORNING, incubated in a CO ₂ incubator (37 ° C., 5% CO ₂ ) for one day, the medium was aspirated off and CHO-S-SFM-II (Invitrogen) 6.9 mL of medium was added. The prepared plasmid was introduced into the cells by the lipofection method. After the obtained culture supernatant was recovered, the cells were removed by centrifugation (about 2000 g, 5 minutes at room temperature), and further sterilized by passing through a 0.22 μm filter MILLEX (R) -GV (Millipore) to obtain a culture supernatant. Antibodies were purified from the obtained culture supernatants using rProtein A Sepharose ^™ Fast Flow (Amersham Biosciences). Purified antibody concentration was measured for absorbance at 280 nm using a spectrophotometer. From the obtained values, the antibody concentration was calculated using the extinction coefficient calculated by the PACE method (Protein Science 1995; 4: 2411-2423).

Measurement of Antibody Plasma Concentration, CRP Concentration, and Unbound soluble IL-6 Receptor by Monkey PK / PD Test

The concentration measurement in cynomolgus monkey plasma was measured by a method known to those skilled in the art by ELISA method. The CRP concentration was measured by Sears R CRP (Kanto Chemical Co., Ltd.), using an automatic analyzer (TBA-120FR, Toshiba Medical Systems, Inc.). The concentration of unbound soluble cynomolgus IL-6 receptor in cynomolgus monkey plasma was measured as follows. 30 μL of the cynomolgus monkey's plasma is added to an appropriate amount of rProtein A Sepharose Fast Flow (GE Healthcare) resin dried in a 0.22 μm filter cup (Millipore), thereby making sure that all IgG-type antibodies in the plasma (Monkey IgG, anti Human IL-6 receptor antibody and antihuman IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex) were adsorbed to Protein A. Thereafter, the resultant was spun down with a high speed centrifuge to recover the pass solution. Since the pass solution does not contain the anti-human IL-6 receptor antibody-soluble cynomolgus IL-6 receptor complex bound to protein A, by measuring the concentration of the soluble cynomolgus IL-6 receptor in the protein A pass solution, Unbound soluble IL-6 receptor concentration can be measured. The soluble cynomolgus monkey IL-6 receptor concentration is a standard known to those skilled in the art for measuring human IL-6 receptor concentration using the soluble cynomolgus monkey IL-6 receptor (cIL-6R) produced above as a standard. Measured. The unbound soluble IL-6 receptor rate was calculated by the following formula.

Attach an electronic file to a sequence list

Claims (9)

A therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibodies as an active ingredient;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence set forth in CDR3),
(b) CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3), and amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising a CDR3 having, or
(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 A heavy chain comprising CDR3 having the amino acid sequence of CDR3), and a CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), a CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3), sequence An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in No. 6 (CDR3 of VL3). A therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibodies as an active ingredient;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3), or
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising. A therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease containing the following antibodies as an active ingredient;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3), or
(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3). A method for use in the manufacture of a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease of the following antibodies;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence set forth in CDR3),
(b) CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3), and amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising a CDR3 having, or
(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 A heavy chain comprising CDR3 having the amino acid sequence of CDR3), and a CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), a CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3), sequence An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in No. 6 (CDR3 of VL3). A method for use in the manufacture of a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease of the following antibodies;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3), or
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising. A method for use in the manufacture of a therapeutic agent for arthritis, pediatric chronic arthritis or Castleman's disease of the following antibodies;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3), or
(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3). The following antibodies for use in methods of treating arthritis, pediatric chronic arthritis or Castleman's disease;
(a) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 An antibody comprising a heavy chain comprising CDR3 having the amino acid sequence set forth in CDR3),
(b) CDR1 having the amino acid sequence set forth in SEQ ID NO: 4 (CDR1 of VL3), CDR2 having the amino acid sequence set forth in SEQ ID NO: 5 (CDR2 of VL3), and amino acid sequence set forth in SEQ ID NO: 6 (CDR3 of VL3) An antibody comprising a light chain comprising a CDR3 having, or
(c) CDR1 having the amino acid sequence set forth in SEQ ID NO: 1 (CDR1 of VH3-M73), CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 (CDR2 of VH3-M73), SEQ ID NO: 3 of VH3-M73 A heavy chain comprising CDR3 having the amino acid sequence of CDR3), and a CDR1 having the amino acid sequence of SEQ ID NO: 4 (CDR1 of VL3), a CDR2 having the amino acid sequence of SEQ ID NO: 5 (CDR2 of VL3), sequence An antibody comprising a light chain comprising a CDR3 having the amino acid sequence set forth in No. 6 (CDR3 of VL3). The following antibodies for use in methods of treating arthritis, pediatric chronic arthritis or Castleman's disease;
(a) an antibody comprising a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73),
(b) an antibody comprising a light chain comprising a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3), or
(c) a heavy chain comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 (variable region of VH3-M73), and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8 (variable region of VL3); An antibody comprising a light chain comprising. The following antibodies for use in methods of treating arthritis, pediatric chronic arthritis or Castleman's disease;
(a) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73),
(b) an antibody comprising a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3), or
(c) an antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 (VH3-M73), and a light chain having the amino acid sequence set forth in SEQ ID NO: 10 (VL3).

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